1. Field of the Invention
The invention relates to (1) a method for the detection and/or determination of an antigen specific immunoglobulin, to (2) a novel reagent and to (3) test kits for use in such determinations.
2. Description of the Prior Art, and Other Information
The immunoglobulins can be subdivided into five classes G, A, M, D and E. Immunoglobulins of these classes are indicated with IgG, IgA, IgM, IgD, and IgE respectively. In the following, immunoglobulins of a certain class will be indicated with IgX, in which X means, A, M, D and E for purposes of this invention.
Immunoglobulins are structurally related and contain at least two heavy chains (H-chains) and two light chains (L-chains), which are mutually connected by disulphide bridges and sometimes via additional polypeptides. The heavy and light chains each have a variable and a constant region. Some immunoglobulins consist of a multiple of the basic structure of two heavy and two light chains.
Antibodies are immunoglobulins, which can bind antigens specifically. The antibody specificity is located in the variable regions of the 4 peptide chains, which are all situated at the same side of the molecule (N-terminal side). A number of biological actions of a certain antibody is mediated through the constant regions of the chains of the immunoglobulin molecule concerned.
Antibodies can be cleaved into fragments, which still have antigen specifity and crystallizable fragments without this antigen binding property. Antigen binding fragments are e.g. Fab-, Fab'- and F(ab').sub.2 -fragments. Crystallizable fragments without antigen specifity are e.g. Fc- and Fc'-fragments.
The concentration of immunoglobulins of the several classes in normal human serum is: IgG 8-16 mg/ml, IgA 1.4-4 mg/ml, IgM 0.5-2.0 mg/ml, IgD 0.0-0.4 mg/ml and IgE 0.000017-0.000450 mg/ml.
Quantitatively antibodies of the IgG class constitute therefore the most important group of antibodies. Antibodies of the IgM class are present in the early stages of an infection, so that determination of antibodies of the IgM class is very important for the early detection of an infectious disease.
Antibodies of the IgA, IgD and IgE classes can be present in serum in increased concentration in certain pathological conditions. For example, antibodies of the IgE class are present in increased concentrations in allergic conditions and antibodies of the IgD class are involved in auto-immune diseases.
The determination of antigen specific immuno-globulins of a peculiar class is of particular clinical significance. Antigen specific immunoglobulins can be determined with immunochemical techniques, in which use is made of an immuno component with binding affinity to the antibody to be detected and/or determined. According to a known technique, an immuno component with binding affinity to the antibody to be determined is made insoluble by coupling to a solid carrier and another specific bindable substance is labelled, for example with a fluorescent, chromophoric or radio-active group or with an enzyme. A disadvantage of these techniques is, that if the rheumatoid factor (RF) is present, which is often present in serum, false positive reactions may be obtained.
Furthermore, the methods for separation of immunoglobulins of different classes and especially of antigen specific immunoglobulins of different classes are elaborate and time consuming and give generally only qualitative or semi-quantitative results. Examples of these methods are immuno-diffusion, immuno-electrophoresis and sucrose density gradient centrifugation.
The rheumatoid factor which often causes false-positive results, is itself also an immunoglobulin, usually of the IgM class. The rheumatoid factor (RF) has affinity for antibodies of the IgG class. RF binds via the constant regions of the heavy chains of the IgG molecule and especially that part, which upon cleavage of the antibody is separated from the antigen binding fragments. Because the rheumatoid factor is usually of the IgM class, it will also be bound by anti-IgM immunoglobulins.